PF358 FUNCTIONAL HIERARCHY AMONG PRO‐SURVIVAL BCL‐2 MEMBERS IN DETERMINING RESISTANCE TO VENETOCLAX IN CLL

Background: The Bcl‐2 inhibitor Venetoclax rapidly induces apoptosis in chronic lymphocytic leukemia (CLL) cells in peripheral blood (PB) whereas lymph node (LN) responses are less complete. This may be linked to pro‐survival signals in the LN, where micro‐environmental signals strongly induce Bcl‐XL, Mcl‐1 and Bfl‐1 which are not targeted by Venetoclax. In some patients resistance occurs over time which sometimes can be explained by acquired mutations, but in most cases the mechanism(s) remain unknown. Aims: We hypothesize that changes in expression or shifts in functional interactions among Bcl‐2 family members are key to clinical response and refractory disease. Methods: Intracellular FACS staining for Bcl‐2, Mcl‐1 and Bcl‐XL in circulating recent LN emigrants (CD19 + /CXCR4 high /CD5 dim ) versus returning (CD19 + /CXCR4l low /CD5 high ) lymphocytes in patient samples treated with Venetoclax. To investigate the functional impact of expression of Bcl‐2 family members, profiling with additional BH3 mimetics against Bcl‐XL (A1331852) and Mcl‐1 (S63845) was applied in a CLL LN model. Primary CLL cells were co‐cultured with fibroblasts expressing CD40L in order to mimic T cell help in the LN. Results: Under Venetoclax treatment, levels of Bcl‐2 itself and other family members may increase in some patients, early in clinical response. During short‐term in vitro incubation with Venetoclax or other BH3 mimetics, we established that Mcl‐1 and Bfl‐1 can increase but not Bcl‐2 or Bcl‐XL. The effect on Mcl‐1 and Bfl‐1 can be explained by a stabilization of these normally short‐lived proteins. In vitro CD40 activation renders CLL cells fully resistant to single Venetoclax treatment. Using this model, we find that dual or triple BH3 mimetic combinations targeting Bcl‐2, Bcl‐XL and Mcl‐1 restored killing. Specifically, whereas the IC50 of dual Bcl‐2+Mcl‐1 targeting is >1uM, combined inhibition of Bcl‐2+Bcl‐XL has an IC50 around 0.3uM. Bcl‐2 and Bcl‐XL thus seem to be the most dominant players. However, even when Bcl‐2, Bcl‐XL and Mcl‐1 were inhibited using triple BH3 mimetic treatment, Venetoclax resistance was not fully reversed to unstimulated control (IC50 1 versus 10 nM), highlighting a potential role for Bfl‐1. To further dissect the functional roles, Bim co‐immunoprecipitation was performed. In PB CLL cells, Bim only binds to Bcl‐2 and upon Venetoclax treatment it is released and targets Bax/Bak to kills cells. After CD40 stimulation, Bim shifts predominantly to Bcl‐XL and less to Mcl‐1. Only when applying triple BH3 mimetic combinations targeting Bcl‐2, Bcl‐XL and Mcl‐1, Bim starts to bind Bfl‐1. These observations suggest a hierarchical model in which Bcl‐2 is most important and where upon Venetoclax treatment, Bcl‐XL and Mcl‐1 come into play. When all of these are targeted by triple BH3 mimetics, Bfl‐1 is engaged to intercept apoptosis. Summary/Conclusion: We applied BH3 mimetics in primary CLL and showed that combined targeting of Bcl‐2 members can abrogate resistance to Venetoclax. Moreover, we dissected the roles of the different Bcl‐2 family members and constructed a hierarchical model of Bcl‐2 family members, providing new insights into Venetoclax resistance which may have clinical utility.